CN102137936A - Particles for detecting intracellular targets - Google Patents

Abstract

The present invention provides methods describing the use of nanoparticles modified with binding moieties.

Description

Particles that detect intracellular targets

funding disclosure

This invention was made with government support under grant IU54-CA 119341 from the Cancer Center for Nanotechnology Excellence (NCI/CCNE), award 5DPIOD000285 from the NIH Director's Pioneer Award, and grant EEC-0647560 from the NSEC award. The government has certain rights in this invention.

technical field

The present invention relates to a method for detecting the concentration of a target molecule in a cell using nanoparticles, wherein the nanoparticle includes a binding moiety that can specifically bind the target molecule, and wherein the linkage results in a detectable concentration of the target molecule after binding the target molecule. measurable change.

Background technique

Labeled oligonucleotides are widely used as probes for the detection of specific macromolecular targets such as nucleic acids and proteins. Their ability to bind targets with high specificity allows their use in in vitro assays such as polymerase chain reaction (PCR) methods. However, delivering these types of target-specific probes into living cells presents significant challenges because cells are inherently resistant to nucleic acid uptake and contain multiple pathways to remove such exogenous genetic material. Therefore, methods to deliver these substances into cells in a manner that preserves their specific binding properties and fluorescent signal transduction capacity are of great interest.

The discovery and subsequent development of oligonucleotide-nanoparticle conjugates contributed to molecular diagnostics (Elghanian et al., 1997, Science 111:1078-1081; Nam et al., 2003, Science 308:1884-1886) and materials design (Mirkin et al. et al., 1996, Nature 382:607-609; Alivisatos et al., 1996, Nature 382:609-611; Demers et al., 2003, Angew.Chem.Int.Ed.40:3071-3073) provide multiple new opportunities. Recently, oligonucleotide-functionalized nanoparticles have been shown to enter cells and act as antisense agents to control gene expression (Rosi et al., 2006, Science 312: 1027-1030). These "antisense particles" are not simple delivery vehicles (Sandhu et al., 2002, Bioconjugate Chem. 13:3-6; Tkachenko et al., 2003, J.Am.Chem.Soc.125:4700-4701), but individual entities Regulatory and transfection reagents that undergo cellular internalization, resist enzymatic degradation and bind intracellular targets with affinity constants two orders of magnitude greater than free oligonucleotides (Lytton-Jean and Mirkin, 2005, J. Am. Chem. Soc. 127:12754-12755). Furthermore, they can be easily modified with robust (ie highly stable) labeling materials such as locked nucleic acids (Seferos et al., 2007, ChemBioChem 8:1230-1232) and are not toxic under conditions required for gene regulation. Indeed, it has been shown that oligonucleotide-modified gold nanoparticles are readily taken up by cells in large quantities, unlike the absence of oligonucleotides in solution. This property led to the discovery that oligonucleotide-modified gold nanoparticles can be used as agents for intracellular genetic control, where they provide rapid intracellular delivery of DNA and also increase the activity of oligonucleotides in cells based on their synergistic properties. effect. These oligonucleotide-functionalized nanoparticles have been shown to enter various cell types and can be used to introduce high local concentrations of oligonucleotides.

Gold nanoparticles functionalized densely with DNA were also previously shown to bind complementary DNA in a highly cooperative manner, resulting in binding strengths two orders of magnitude higher than those measured for similar DNA strands not linked to gold nanoparticles. This property makes nanoparticles particularly useful for DNA and protein diagnostic assays, in addition to those uses described above.

One class of oligonucleotides of interest are those that can detect a specific target with a recognition sequence. These structural types, if introduced into living cells, are of particular interest for medical diagnostics, drug development, and developmental and molecular biology applications. However, current delivery/transfection strategies lack the characteristics required for their use, such as 1) low toxicity, 2) high cellular uptake, and 3) providing resistance to enzymes that cause false positive signals.

Probes for imaging and detecting intracellular RNA include those used in in situ staining (Femino et al., Science 280:585-590, 1998; Kloosterman et al., Nat. Methods 3:27-29, 2006), molecular beacons ( Tyagi et al., 1996, Nat.Biotechnol.14:303-308; Sokol et al., 1998, Proc.Natl.Acad.Sci.USA 95:11538-11543; Peng et al., 2005, Cancer Res.65:1909-1917; Perlette etc., 2001, Anal.Chem.73:5544-5550; Nitin et al., 2004, Nucleic Acids Res.32:e58), and FRET pair (Santangelo et al., 2004, Nucleic Acids Res.32:e57; Rratu et al., 2003, Proc USA 100: 13308-13313), each of which is an important biological tool for measuring and quantifying the activity of living systems in response to external stimuli (Santangelo et al., 2006, Annals of Biomedical Engineering 34: 39-50). However, the delivery of oligonucleotide-based reporters into the cell matrix and cells has proven to be a formidable challenge for intracellular detection. Cellular internalization of oligonucleotide-based probes usually requires transfection reagents such as lipids (Zabner et al., 1995, J. Bio. Chem. 270:18997-19007) or dendrimers (Kukowska-Latallo et al., 1996, USA 93:4897-4902), which can be toxic or alter cellular processes. Furthermore, oligonucleotides are prone to intracellular degradation (Opalinska and Gewirtz, 2002, Nat. Rev. Drug Disc. 1:503-514), and in the case of fluorophore-labeled probes, this can lead to indistinguishable High background signal for true recognition events (Li et al., 2004, Nucleic Acids Res. 28:e52; Rizzo et al., 2002, Molecular and Cellular Probes 16:277-283).

Therefore, although nanoparticles that can recognize targets with high specificity have been designed, it is difficult to detect positive effects derived from specific interactions, especially at the single-cell level with high sensitivity.

Therefore, there is a need in the art to develop materials that can enter cells to connect to specific targets and methods for detecting and quantifying intracellular interactions.

Contents of the invention

The present invention provides a method of determining the concentration of a target molecule in a cell comprising the step of contacting the target molecule with a nanoparticle comprising a protein specific for the target molecule under conditions that allow the target molecule to attach to the nanoparticle. a binding moiety, the binding moiety is labeled with a label, wherein attachment of the target molecule to the nanoparticle results in a detectable change in the label, and wherein the change in the detectable label is proportional to the concentration of the target molecule in the cell Proportion.

In one embodiment of this method, the binding moiety is a polynucleotide, and in another aspect, the binding moiety is a polypeptide. In embodiments where the binding moiety is a polynucleotide, other aspects include those wherein the binding moiety is a DNA molecule or an RNA molecule. In other embodiments of this method, the target molecule is a polynucleotide or polypeptide. In embodiments where the target molecule is a polynucleotide, other aspects include those wherein the binding moiety is a DNA molecule or an RNA molecule.

In one embodiment, the invention provides a method wherein the binding moiety is a polynucleotide covalently linked to the nanoparticle and the label is a label linked to a polynucleotide that hybridizes to the binding moiety polynucleotide, wherein the binding moiety is polynuclear Ligation of the nucleotide and target molecule releases the hybridized polynucleotide, and the label is detectable after release. In one aspect, a label is attached to the hybridized polynucleotide, and when the hybridized polynucleotide bearing the label hybridizes to the binding moiety, the label is quenched.

In another embodiment, the invention provides methods wherein the binding moiety is a polypeptide covalently linked to the nanoparticle and the label is a label linked to a reagent linked to the binding moiety polypeptide, wherein the binding moiety polypeptide and the target molecule The ligation displaces the attached reagent, and the label is detectable after release. In one aspect, a label is attached to the reagent, and when the reagent is attached to the binding moiety polypeptide, the label is quenched.

In another embodiment, the invention provides methods wherein the binding moiety is labeled with a label, and the label is only detectable when the binding moiety is attached to a target molecule. In embodiments where the binding moiety is a polynucleotide, other aspects include those wherein the binding moiety is a DNA molecule or an RNA molecule. In other embodiments of this method, the target molecule is a polynucleotide or polypeptide. In embodiments where the target molecule is a polynucleotide, other aspects include those wherein the binding moiety is a DNA molecule or an RNA molecule.

In one aspect, the binding moiety is a polynucleotide and the label is attached to the polynucleotide binding moiety such that when the polynucleotide binding moiety is not attached to the target molecule, the label is quenched. Thus, the label attached to the polynucleotide binding moiety can only be detected when the polynucleotide binding moiety is attached to the target molecule.

In another aspect, the binding moiety is a polypeptide and the label is attached to the polypeptide binding moiety such that when the polypeptide binding moiety is not attached to the target molecule, the label is quenched. Thus, the polypeptide-binding moiety can only be detected when it is linked to a target molecule.

The invention also provides methods wherein the nanoparticles comprise a plurality of binding moieties. In one aspect, the invention provides methods wherein multiple binding moieties are specifically linked to one target molecule. In another aspect, wherein multiple binding moieties specifically bind to more than one target molecule.

Other aspects of the invention will be apparent from the following detailed description. It should be understood, however, that the following detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art described in detail. It is obvious.

Description of drawings

Figure 1 provides a diagram of gold nanoparticles modified with fluorophore-containing oligonucleotides capable of detecting intracellular targets.

Figure 2 shows nano-flares for mRNA detection and quantification.

Figure 3 depicts the quantification of survivin knockdown using nano-flares. (a) Flow cytometric data collected on siRNA-treated SKBR3 cells, (b) plot of mean fluorescence (black circles) and survivin expression (grey histogram) as a function of siRNA concentration.

Detailed ways

The methods provided herein take advantage of the physical properties and use of nanoparticles modified to include one or more binding moieties that specifically recognize and link to one or more specific target moieties. It is shown herein that the concentration of a target molecule within a cell can be determined using nanoparticles comprising a binding moiety specific for the target molecule. In the present invention, the binding moiety is labeled with a label, wherein attachment of the target molecule to the nanoparticle results in a detectable change in the label, and wherein the change in the detectable label is proportional to the concentration of the target molecule in the cell. Proportion. It is to be appreciated that intracellular targets include those naturally occurring in the target cell, those naturally occurring targets that have been introduced into the cell (but not normally found in this cell type) or those that are not present but have been introduced into the cell Synthetic targets in target cells.

In another aspect of the invention, the methods outlined herein can also be used to determine the localization of a desired target within a cell.

As used herein, it is understood that the term "binding moiety" encompasses polynucleotides and polypeptides, or any other fragment or portion of the foregoing molecules that can be linked to a target of interest. This term includes, but is not limited to, small molecules of interest. As understood in the art, the term "small molecule" includes organic and inorganic compounds, which are either naturally occurring compounds, modifications of naturally occurring compounds, or synthetic compounds.

The provided methods are particularly suitable for use with binding moieties that recognize and link intracellular target molecules, wherein the binding moieties are polynucleotides and/or polypeptides and the target molecules are polynucleotides and/or polypeptides. In simple aspects, a polynucleotide binding moiety is specifically linked to a polynucleotide target molecule, or a polypeptide binding moiety is specifically linked to a polypeptide target molecule. However, methods are also contemplated wherein the polynucleotide binding moiety is specifically linked to the polypeptide target molecule, or the polypeptide binding moiety is specifically linked to the polynucleotide target molecule.

As used herein, the term "specifically recognizes" or "specifically binds" means that a binding moiety can recognize and/or bind to one target molecule with a higher affinity and/or activity than all other target molecules interaction.

The method provides functionality based on the principle that the binding moiety is directly or indirectly labeled with a label and that attachment of the binding moiety to the target molecule results in the label becoming detectable or more detectable. Thus, when the binding moiety is not attached to the target molecule, the label is relatively undetectable or quenched. While the term "quenching" is understood in the art in general in relation to fluorescent labels, it is contemplated herein that the signal of any label is quenched when it is relatively undetectable. Therefore, it should be understood that the methods listed in this specification for applying fluorescent markers are provided only as one embodiment of the contemplated methods, and that any quenchable markers may be substituted for the exemplary fluorescent markers.

In one aspect, the label is a label directly attached to the binding moiety, and in another aspect, the label is attached to a reagent attached to the binding moiety that has a low binding affinity or activity for the binding moiety such that the target Attachment of a molecule to a binding moiety causes the reagent to be displaced from the binding moiety to which it is attached.

When a label is directly attached to a binding moiety, the label can be positioned such that it is relatively undetectable or quenched when the binding moiety is not attached to a target molecule. For example, for the polynucleotide-binding moiety not attached to the target molecule, the secondary structure formed inside the polynucleotide-binding moiety can localize the label close to the nanoparticle itself, or the label can freely swing in the aqueous environment , so that at any given time the marker can be close to the nanoparticle and its signal quenched, or the marker can swing freely in the aqueous environment away from the nanoparticle (while still attached to the nanoparticle), so that the signal is not quenched. In embodiments where there is no secondary structure to hold the label close to the quenching position of the nanoparticle, it is necessary to detect the level of background signal when the polynucleotide binding moiety is not attached to the target molecule, and the polynucleotide binding moiety is associated with the target molecule. Attachment of the target molecule will cause the signal to rise above background as more label is displaced from the nanoparticle close enough to impart quenching.

Similarly, when the binding moiety is a polypeptide, the label on the binding moiety can be positioned such that a conformational change occurs upon attachment of the polypeptide binding moiety to the target molecule, causing the label to be far enough away from the nanoparticle that its signal is relatively unquenched .

In terms of methods where the label is not directly attached to the binding moiety, the attachment of the binding moiety to the target molecule results in physical release of the target such that the nanoparticles cannot exert a quenching effect on the label. For example, for a polynucleotide binding moiety, a label can be labeled on a second polynucleotide to which the polynucleotide binding moiety hybridizes, in a position such that the label is in sufficient proximity to the nanoparticle for the nanoparticle to exert its quenching effect . When the polynucleotide-binding molecule recognizes and ligates the target molecule, the hybridized and labeled polynucleotide is displaced and quenching of the nanoparticles ceases.

Thus, in one aspect, for example, the present invention provides methods wherein gold nanoparticles modified to contain polynucleotide binding moieties can be used as transfection agents and cellular "nano-flares" for imaging and quantifying RNA in live cells, so The polynucleotide binding moiety in turn hybridizes to a complementary polynucleotide labeled with a fluorophore label. The advantages of nano-flares are the highly efficient fluorescence quenching properties of gold (Dubertret et al., 2001, Nat. Biotechnol. 19:365-370), the cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the The enzymatic stability of the conjugate (Rosi et al., 2006, Science 312: 1027-1030), therefore overcomes many challenges to generate sensitive and efficient intracellular probes. In particular, nano-flares display high signaling, have low background fluorescence, and are sensitive to changes in the number of intracellular RNA transcripts. Thus, the nano-flares described herein are oligonucleotide-functionalized nanoparticle conjugates designed to provide intracellular fluorescent signals that correlate directly or indirectly with the relative amount of specific intracellular RNA. RNAs that it is desirable to detect in the disclosed methods include, but are not limited to, mRNA and hnRNA.

A similar mechanism applies to polypeptide-binding moieties, where the reagent labeled with a label and reagent can be linked to the polypeptide-binding moiety such that its attachment brings the kit label into sufficient proximity to the nanoparticle for relative quenching of the label. When the polypeptide binding moiety is attached to a specific target molecule, the nano-flare agent as described above is released or displaced, and the quenching effect of the nanoparticle is terminated.

Regardless of the specific nature of the binding moiety, some of the difficulties normally associated with intracellular molecular detection can be alleviated by using fluorophore-labeled oligonucleotides or polypeptides to densely functionalize nanoparticles. These binding moieties do not require microinjection or co-transfection agents to enter cells, are highly resistant to enzymatic degradation, and are nontoxic under the conditions studied.

polynucleotide

As used herein, the term "polynucleotide", either functionalized on a nanoparticle or as a targeting molecule, may be used interchangeably with the term oligonucleotide.

As used herein, the term "nucleotide" is used interchangeably with modified forms described herein and known in the art. In certain instances, the term "nucleobase" is used in the art to encompass naturally occurring nucleotides as well as modifications of nucleotides that can be polymerized.

Methods for preparing polynucleotides of predetermined sequence are well known in the art. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid phase synthesis methods are preferred for oligoribonucleotides and oligodeoxyribonucleotides (well known methods for the synthesis of DNA can also be used for the synthesis of RNA). Oligoribonucleotides and oligodeoxyribonucleotides can also be prepared enzymatically. The synthesis of oligoribonucleotides and oligodeoxyribonucleotides is preferably carried out by solid phase synthesis (well known methods for the synthesis of DNA are also used for the synthesis of RNA). Oligoribonucleotides and oligodeoxyribonucleotides can also be prepared enzymatically.

In various inventions, the methods provided herein include the use of polynucleotides that are DNA oligonucleotides, RNA oligonucleotides, or a combination of these two types. Modified forms of oligonucleotides are also contemplated for use, including those having at least one modified internucleotide linkage. Modified polynucleotides or oligonucleotides are described in detail below.

modified oligonucleotides

Specific examples of oligonucleotides include those containing modified backbones or non-natural internucleotide linkages. Oligonucleotides with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides having no phosphorus atoms in the internucleotide backbone are encompassed within the meaning of "oligonucleotide".

Modified oligonucleotide backbones containing phosphorus atoms include, for example, phosphorothioate with normal 3'-5' linkage, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, Methyl and other alkyl phosphonates, phosphinates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, including 3'-aminophosphoramidates and aminoalkylamino Phosphonate phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, phosphoroselenophosphates and boranophosphates; 2'-5' linked analogs thereof; and A backbone in which one or more of the internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage is reversed in polarity. Also contemplated are oligonucleotides with inverted polarity that include a pure 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single inverted nucleotide residue, which may be abasic (the nucleotide is missing or has a hydroxyl group in its place). Salt, mixed salt and free acid forms are also possible.教导上述含磷连接的制备的美国专利包括，美国专利3,687,808；4,469,863；4,476,301；5,023,243；5,177,196；5,188,897；5,264,423；5,276,019；5,278,302；5,286,717；5,321,131；5,399,676；5,405,939；5,453,496；5,455,233；5,466,677；5,476,925；5,519,126； 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;

Modified oligonucleotide backbones that do not include phosphorus atoms have internucleotide linkages through short chain alkyl or cycloalkyl, mixed heteroatom and alkyl or cycloalkyl internucleotide linkages, or one or more A backbone formed by bonds between short-chain heteroatoms or heterocyclic nucleotides. These backbones include those with morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; olefin-containing backbones; sulfamate backbones; Methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and _CH2 moieties.参见，例如，美国专利5,034,506；5,166,315；5,185,444；5,214,134；5,216,141；5,235,033；5,264,562；5,264,564；5,405,938；5,434,257；5,466,677；5,470,967；5,489,677；5,541,307；5,561,225；5,596,086；5,602,240；5,610,289；5,602,240；5,608,046；5,610,289；5,618,704； 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, the disclosures of which are hereby incorporated by reference in their entirety.

In yet another embodiment, oligonucleotide mimetics wherein one or more sugars and/or one or more internucleotide linkages in the nucleotide unit are replaced by "non-naturally occurring" groups . In one aspect, this embodiment relates to peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by an amide-containing backbone. See, eg, US Patents 5,539,082; 5,714,331; and 5,719,262, and Nielsen et al, 1991, Science, 254:1497-1500, the disclosures of which are incorporated herein by reference.

In yet another embodiment, there is provided an oligonucleotide having a phosphorothioate backbone and having a heteroatom backbone and comprising -CH ₂ -NH-O-CH ₂ -, -CH ₂ -N(CH ₃ )-O-CH ₂ -, -CH ₂ -ON(CH ₃ )-CH ₂ -, -CH ₂ -N(CH3)-N(CH ₃ )-CH ₂ - and -ON(CH ₃ )-CH ₂ -CH ₂ - Oligonucleotides (as described in US Patent Nos. 5,489,677 and 5,602,240). Oligonucleotides having a morpholino backbone structure are also provided, as described in US Patent No. 5,034,506.

In various forms, the link between two consecutive monomers in the oligomer consists of 2-4, preferably 3 groups/atoms selected from -CH ₂ -, -O-, -S-, -NR ^H -, >C=O, >C=NR ^H , >C=S, -Si(R″) ₂ -, -SO-, -S(O) ₂ -, -P(O ) ₂ -, -PO(BH ₃ )-, -P(O, S)-, -P(S) ₂ -, -PO(R″)-, -PO(OCH ₃ )- and -PO(NHR ^H )-, wherein R ^H is selected from hydrogen and C _1-4 alkyl; R″ is selected from C _1-6 alkyl and phenyl. Illustrative examples of these linkages are -CH ₂ -CH ₂ -CH ₂ -, -CH ₂ -CO-CH ₂ -, -CH ₂ -CHOH-CH ₂ -, -O-CH ₂ -O-, -O-CH ₂ -CH ₂ -, -O-CH ₂ -CH=(when used Including R ⁵ ), -CH ₂ -CH ₂ -O-, -NR ^H -CH 2 -CH 2 -, -CH ₂ -CH ₂ -NR _H ^- , -CH ₂ -NR when used as _a link to a subsequent monomer ^H -CH ₂ -, -O-CH ₂ -CH ₂ -NR ^H -, -NR ^H -CO-O-, -NR ^H -CO-NR ^H -, -NR ^H -CS-NR ^H -, -NR ^H -C(=NR ^H )-NR ^H -, -NR ^H -CO-CH ₂ -NR ^H -O-CO-O-, -O-CO-CH ₂ -O-, -O-CH ₂ -CO -O-, -CH ₂ -CO-NR ^H -, -O-CO-NR ^H -, -NR ^H -CO-CH ₂ -, -O-CH ₂ -CO-NR ^H -, -O-CH ₂ -CH ₂ -NR ^H -, -CH=NO-, -CH ₂ -NR ^H -O-, -CH ₂ -ON= (including R ⁵ when used as a link to a subsequent monomer), -CH ₂ - O-NR ^H -, -CO-NR ^H -CH ₂ -, -CH ₂ -NR ^H -O-, -CH ₂ -NR ^H -CO-, -O-NR ^H -CH ₂ -, -O-NR ^H , -O-CH ₂ -S-, -S-CH ₂ -O-, -CH ₂ -CH ₂ -S-, -O-CH ₂ -CH ₂ -S-, -S-CH ₂ -CH= (including ^R5 when used as a link to a subsequent monomer), -S- _CH2 - _CH2- , -S-CH2 _-CH2 -O-, -S-CH2-CH2-S-, -CH2- S- _CH2- , _-CH2- SO- _CH2- , -CH2-SO2-CH ₂ -, -O-SO -O-, -OS(O)2-O-, -OS(O) ₂ - CH ₂ -, -OS(O) ₂ -NR ^H -, -NR ^H -S(O ) ₂ -CH ₂ -; -OS(O) ₂ -CH2-, -OP(O) ₂ -O-, -OP(O, S)-O-, -OP(S) ₂ -O-, -SP (O) ₂ -O-, -SP(O, S)-O-, -SP(S) ₂ -O-, -OP(O) ₂ -S-, -OP(O ₅ S)-S-, -OP(S) ₂ -S-, -SP(O) ₂ -S-, -SP(O, S)-S-, -SP(S) ₂ -S-, -O-PO(R″)- O-, -O-PO(OCH ₃ )-O-, -0-PO(O CH ₂ CH ₃ )-O-, -O-PO(O CH ₂ CH ₂ SR)-O-, -O-PO (BH ₃ )-O-, -O-PO(NHR ^N )-O-, -OP(O)2-NR ^H H-, -NR ^H -P(O) ₂ -O-, -OP(O, NR ^H )-O-, -CH ₂ -P(O) ₂ -O-, -O-P(O) ₂ -CH2-, and -O-Si(R″) ₂ -O-; where, considering- CH ₂ -CO-NR ^H -, -CH2-NR ^H -O-, -S-CH2-O-, -OP(O)2-OOP(-O, S)-O-, -OP(S) ₂ -O-, -NR ^H P(O) ₂ -O-, -OP(O, NR ^H )-O-, -O-PO(R″)-O-, -O-PO(CH ₃ )-O - and -O-PO( ^NHRN )-O-, wherein R ^H is selected from hydrogen and C _1-4 alkyl, and R″ is selected from C _1-6 alkyl and phenyl. Further illustrative examples are listed in Mesmaeker et. al, 1995, Current Opinion in Structural Biology, 5: 343-355 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol 25: pp 4429-4443 .

Additional modified forms of oligonucleotides are described in detail in US Patent Application No. 20040219565, the disclosure of which is hereby incorporated by reference in its entirety.

Modified oligonucleotides may also contain one or more substituted sugar moieties. In certain aspects, the oligonucleotide comprises one of the following groups at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O -, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and Alkynyl. Other embodiments include O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH 2 ) _n NH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ ) _n ONH ₂ and O(CH2)nON[( _CH2 ) _nCH3 ] ₂ , where n and m range _from 1 to about 10. Other oligonucleotides contain one of the following groups at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, Polyamino amino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties of oligonucleotides, or groups that improve the efficacy of oligonucleotides, and other substituents with similar properties. In one aspect, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethoxy) or 2'-MOE) (Martin et al. al, 1995, HeIv.CMm.Acta, 78:486-504) ie, alkoxyalkoxy groups. Other modifications include 2'-dimethylaminooxyethoxy, ie, the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples, and 2'- Dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy- and or 2'-DMAEOE), ie, 2'-O-CH2-O- CH2-N(CH3)2, is also described in the following examples.

Other modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2 '-O-allyl (2'-O-CH2-CH=CH2) and 2'-fluoro (2'-F). The 2' modification can be at the arabino (upper) position or the ribo (lower) position, in one aspect the 2'-arabino modification is 2'-F. Similar modifications can also be made on the oligonucleotide, for example at the 3' position of the sugar in the 3' terminal nucleotide or in a 2'-5' linked oligonucleotide, and at the 5' position of the 5' terminal nucleotide. 'Location. Oligonucleotides may also have sugar analogs, for example cyclobutyl moieties substituted for pentofuranosyl sugars.参见，例如美国专利4,981,957；5,118,800；5,319,080；5,359,044；5,393,878；5,446,137；5,466,786；5,514,785；5,519,134；5,567,811；5,576,427；5,591,722；5,597,909；5,610,300；5,627,053；5,639,873；5,646,265；5,658,873；5,670,633；5,792,747；和5,700,920，其公开Incorporated herein by reference in its entirety.

In one aspect, sugar modifications include Locked Nucleic Acids (LNAs), in which the 2'-hydroxyl group is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is in some aspects a methylene (-CH2-)n group, bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and their preparation are described in WO 98/39352 and WO 99/14226.

Oligonucleotides may also include base modifications or substitutions, "unmodified" or "natural" bases as used herein include the purine bases adenine (A) and guanine (G), and the pyrimidine base thymine (T), cytosine (C) and uracil (U). Modified bases include other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6 - Methyl and other alkyl substituted adenine and guanine, 2-propyl and other alkyl substituted adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytidine Pyrimidine, 5-halouracil and cytosine, 5-propyneuracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil ( pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo Generation, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methylguanine, 2-F-adenine, 2 -Amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine (deazaguanine) and 3-deazaadenine (deazaadenine ). Further modified bases include tricyclic pyrimidines, such as phenoxazincytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenoxazincytidine Glycosides (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g. 9-(2-amino Ethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolidine (2H-pyrimido[4,5-b] indol-2-one), pyrimidine indolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may also include those in which purine or pyrimidine bases are replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine and 2-pyridone. Additional bases include those disclosed in U.S. Patent No. 3,687,808, disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.L, ed. John Wiley & Sons, 1990, disclosed in Englisch et al. al, 1991, Angewandte Chemie, International Edition, 30:613 and bases in Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993. Some of these bases are used to increase affinity, including 5-substituted pyrimidines, 6-azopyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propyne uracil and 5-propyne cytosine. 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2°C and, in some aspects, in combination with 2'-O-methoxyethyl sugar modifications.参见，例如美国专利3,687,808，美国专利4,845,205；5,130,302；5,134,066；5,175,273；5,367,066；5,432,272；5,457,187；5,459,255；5,484,908；5,502,177；5,525,711；5,552,540；5,587,469；5,594,121，5,596,091；5,614,617；5,645,985；5,830,653；5,763,588；6,005,096；5,750,692 and 5,681,941, the disclosures of which are incorporated herein by reference.

"Modified bases" or other antecedents refer to compositions capable of pairing with natural bases (e.g., adenine, guanine, cytosine, uracil, and/or thymine), and/or capable of pairing with non-naturally occurring base pairing composition. In certain aspects, the modified base provides a Tm difference of 15, 12, 10, 8, 6, 4, or 2°C. Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.

"Nucleic acid base" means the naturally occurring nucleic acid bases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleic acid bases groups, such as xanthine, diaminopurine, 8-oxo-N ⁶ -methyladenine, 7-azaxanthine, 7-azazaguanine, N ⁴ , N ⁴ -ethoxycytosine, N',N'-ethoxy-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromourea Pyrimidine, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyrimidine, isocytosine, isoguanine, inosine and in Benner et al, USPat.No.5,432,272 and Susan M.Freier and Karl - "Non-naturally occurring" nucleic acid bases disclosed in Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. Therefore, the term "nucleic acid base" includes not only the known purine and pyrimidine heterocycles, but also their heterocycle analogs and tautomers. Further, naturally and non-naturally occurring nucleic acid bases are included in USPat.No.3,687,808 (Merigan, et al), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed.STCrooke and B.Lebleu, CRC Press, 1993, in Englisch et al, 1991, Angewandte Chemie, International Edition, 30:613-722 (see pages 622 and 623 for details, and Concise Encyclopedia of Polymer Science and Engineering, JI Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Nucleic acid bases disclosed in Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which is incorporated herein by reference). The terms "nucleotide base" and "base unit" further include compounds such as heterocyclic compounds which can be used as nucleotides, including certain "universal bases" which are not traditionally nucleotide bases but which function as The role of nucleotide bases. A general base specifically mentioned is 3-nitropyrrole, which may be substituted by indoles (eg, 5-nitroindole), and may also be substituted by hypoxanthine. Additional desirable universal bases include pyrrole, oxadiazole or triazole derivatives, including universal bases known in the art.

polypeptide

As used herein, "polypeptide" refers to naturally occurring, synthetically produced or recombinantly produced peptides, proteins, amino acid polymers, hormones, viruses and antibodies. Polypeptides also include lipoproteins and post-translationally modified proteins, e.g., glycosylated proteins, as well as having D-amino acids in their D- or L-configuration and/or peptidomimetic units as part of their structure, modified, derivatized or A protein or proteinaceous substance that is not a naturally occurring amino acid.

Peptides for use in the methods of the invention include peptides from commercially available sources. Peptide libraries include: (i) structured peptide libraries comprising small, disulfide bond-constrained cyclic peptide compounds ranging in size from six to twelve amino acids, where the number of distinct peptide structures in each library exceeds one billion; (ii) A linear peptide library in which 19 amino acids (without cysteine) at each position in a 20-mer peptide allows the formation of a library of 10 billion peptides; (iii) a substrate phage peptide library in which in a 13-mer peptide All 19 amino acids at each position allow the formation of a library of approximately 100 billion peptides.

Commercially available peptide libraries include those from Peptide libraries Eurogentec s.a. (Belgium), Dyax Corp. (Cambridge, MA) and Cambridge Peptide (Cambridge, UK).

The preparation of peptide libraries for the implementation of the methods of the present invention is well known in the art, as described in Jung (ed) Combinatorial Peptide and Nonpeptide Libraries: A Handbook and in Devlin et al, 1990, Science, VoI 249, Issue 4967: 404-406, It can also be prepared by utilizing commercially available synthesis kits, such as those available from Sigma-Genosys.

Proteins useful in the methods of the present invention include those derived from synthetic protein libraries (recorded in Matsuura, et al, 2002, Protein Science 11: 2631-2643, Ohuchi et al., 1998, Nucleic Acids Res. October 1; 26(19 ): 4339-4346, WO/1999/011655, WO/1998/047343, US Patent No. 6844161 and US Patent No. 6403312). Commercially available kits for producing protein libraries are also known in the art and can be purchased, for example, from BioCat GmbH (Heidelberg).

Protein libraries useful in the practice of the methods of the invention are also commercially available, for example from Dyax Corporation (Cambridge, MA).

Detectable markers/markers

The term "label" is used herein interchangeably with "label" regardless of the type of identified compound interacting, and the present invention provides methods wherein polynucleotide or polypeptide complex formation is visually altered. Detected. In one aspect, complex formation provides a color change that can be observed with the naked eye or spectroscopically. When using gold nanoparticles, a red to blue change occurs as the nanoparticles aggregate, often observed with the naked eye. In the present invention, said aggregation is considered to occur as a result of the binding of separate nanoparticles to the same target molecule, each separate nanoparticle containing a binding moiety bound to a specific but located different part of the target molecule.

In another aspect, polynucleotide or polypeptide complex formation causes aggregation to occur, which can be visualized by electron microscopy or nephelometry. In general, aggregation of nanoparticles leads to a decrease in plasmon resonance. In yet another aspect, complex formation results in the precipitation of aggregated nanoparticles, which can be observed with the naked eye or a microscope.

In one aspect, observation of the color change with the naked eye is performed against a background of a contrasting color. For example, when gold nanoparticles are used, observation of color changes is facilitated by spotting a sample of the hybridization solution onto a solid white surface (such as, but not limited to, silica or alumina TLC plates, filter paper, nitrocellulose membranes, nylon membranes, or C-18 silica TLC plate), and then dry the spot. Initially, the spot retains the color of the hybridization solution, which is roughly pink/red when not hybridized, and purplish-red/purple when hybridized. When dried at room temperature or 80°C (temperature is not critical), blue dots are formed if the nanoparticle-oligonucleotide conjugates have been linked by hybridization prior to spotting. When not hybridized, the dots are pink. The blue and pink dots are stable and do not change on subsequent cooling or heating, nor over time, providing a convenient permanent record of the assay. Additional steps, such as steps to separate hybridized and unhybridized nanoparticle-oligonucleotide conjugates, are not necessary to observe this color change.

An alternative method for visualizing the results of the practice of the invention is to spot samples of nanoparticle probes onto membrane fiber filters (e.g., borosilicate microfiber filters, 0.7 micron pore size, FG75 grade, for 13 nm sized gold nanoparticles) while pumping liquid through the filter. Excess unhybridized probe is then rinsed through the filter, leaving observable spots containing aggregates due to hybridization of the nanoparticle probes (since these aggregates are larger than the pores of the filter). This technique allows for greater sensitivity since an excess of nanoparticles can be used.

It is understood that the labels include any of the fluorophores described herein as well as other detectable labels known in the art. For example, labels can also include, but are not limited to, redoxactive probes, other nanoparticles, and quantum dots, as well as any label that can be detected spectroscopically, i.e., using microscopy and blood cell counting. of markers.

Methods for labeling oligonucleotides

Methods of labeling oligonucleotides with fluorescent molecules and measuring fluorescence are known in the art. Suitable fluorescent molecules are also known in the art and include, but are not limited to: 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid), 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS ), 5-(and-6)-carboxy-2′,7′-dichlorofluorescein pH 9.0, 5-FAMP pH 9.0, 5-ROX (5-carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA, 5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6 JOE, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-Carboxyrhodan Ming 6G pH 7.0, 6-Carboxyrhodamine 6G, Hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SE pH 9.0, 7-Amino-4-methylcoumarin pH 7.0, 7-Hydroxy-4 -Methylcoumarin, 7-Hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa647, Alexa 660, Alexa 680, Alexa 700, Alexa Fluor 430 Antibody Conjugate pH 7.2, Alexa Fluor 488 Antibody Conjugate pH 8.0, Alexa Fluor 488 hydrazide-water, Alexa Fluor 532 Antibody Conjugate pH 7.2, Alexa Fluor 555 Antibody Conjugate Conjugate pH 7.2, Alexa Fluor568 Antibody Conjugate pH 7.2, Alexa Fluor 610R-Phycoerythrin Streptomycin pH 7.2, AlexaFluor 647 Antibody Conjugate pH 7.2, Alexa Fluor 647 R-Phycoerythrin Streptomycin pH 7.2, Alexa Fluor 660 Antibody Conjugate pH 7.2, Alexa Fluor 680 Antibody Conjugate pH 7.2, AlexaFluor 700 Antibody Conjugate pH 7.2, AIlophycocyanin pH 7.5, AMCA Conjugate, Aminocoumarin, APC (Allophycocyanin) , Atto 647, BCECF pH 5.5, BCECF pH 9.0, BFP (Blue Fluorescent Protein), BO-PRO-I-DNA, BO-PRO-3-DNA, BOBO-I-DNA, BOBO-3-DNA, BODIPY 650 /665-X, MeOH, BODIPY FL Conjugate, BODIPYFL, MeOH, Bodipy R6G SE, BODIP Y R6G, MeOH, BODIPY TMR-X Antibody Conjugate pH 7.2, Bodipy TMR-X Conjugate, BODIPY TMR-X, MeOH, BODIPYTMR-X, SE, BODIPY TR-X-like Phalloidin pH 7.0, BODIPY TR-X, MeOH, BODIPY TR-X, SE, BOPRO-I, BOPRO-3, Calcein, Calcein pH 9.0, Calcium Crimson, Calcium Crimson Ca ²⁺ , Calcium Green, Calcium Green-1 Ca ²⁺ , Calcium Orange, Calcium Orange Ca ²⁺ , Carboxynaphthofluorescein pH 10.0, Cascade Blue, Cascade Blue BSA pH 7.0, Cascade Yellow, Cascade Yellow Antibody Conjugate pH 8.0, CFDA, CFP (cyanofluorescent protein), CI- NERF pH 2.5, CI-NERF pH 6.0, Tartrazine, Coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, CyQUANTGR-DNA, Dansylcadaverine, Dansylcadaverine, MeOH, DAPI, DAPI-DNA, Dapoxyl(2-aminoethyl)sulfonamide, DDAO pH 9.0, Di-8 ANEPPS, Di-8-ANEPPS-Lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed , DTAF, dTomato, eCFP (enhanced cyanofluorescent protein), eGFP (enhanced green fluorescent protein), Eosin, Eosin antibody conjugate pH 8.0, erythrosine-5-isothiocyanate pH 9.0, ethyl bromide Met, Humidimer Homodimer, Hoomiamine Homodimer-1-DNA, eYFP (Enhanced Yellow Fluorescent Protein), FDA, FITC, FITC Antibody Conjugate pH 8.0, FlAsH, Fluo-3, Fluo -3Ca2+, Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1M NaOH, Fluorescein Antibody Conjugate pH 8.0, Fluorescein Dextran pH 8.0, Fluorescein pH 9.0, Fluoro-Emerald, FM 1-43 , FM 1-43 fat, FM 4-64, FM 4-64, 2% CHAPS, Fura red Ca ²⁺ , Fura red, high Ca, Fura red, low Ca, Fura-2 Ca ²⁺ , Fura-2, High Ca, Fura-2, Ca-free, GFP(S65T), HcRed, Hoechst 33258, Hoechst33258-DNA, Hoechst 33342, Indo-1 Ca ²⁺ , Indo-1, Ca-free, Indo-1, Ca saturated, JC- I, JC-I pH 8.2, Lissar nine rhodamine, LOLO-I-DNA, fluorescent yellow, CH, LysoSensor blue, LysoSensor blue pH 5.0, LysoSensor green, LysoSensor green pH 5.0, LysoSensor yellow pH 3.0, LysoSensor yellow pH 9.0, LysoTracker blue, LysoTracker green, LysoTracker red, Magnesium Green, Magnesium Green Mg ²⁺ , Magnesium Orange, Marina Blue, mBanana, mCherry, mHoneydew, MitoTracker Green, MitoTracker Green FM, MeOH, Mito Tracker Orange, MitoTracker Orange, MeOH, MitoTracker Red, MitoTracker Red, MeOH, mOrange, mPlum , mRFP, mStrawberry, mTangerine, NBD-X, NBD-X, MeOH, NeuroTrace500/525, green fluorescent Nissl stained RNA, Nile blue, EtOH, Nile red, Nile red-lipid, Nissl, Oregon green 488, Oregon green 488 Antibody Conjugate pH 8.0, Oregon Green 514, Oregon Green 514 Antibody Conjugate pH 8.0, Pacific Blue, Pacific Blue Antibody Conjugate pH 8.0, Phycoerythrin, PicoGreen dsDNA Quantification Reagent, PO-PRO-I, PO-PRO- I-DNA, PO-PRO-3, PO-PRO-3-DNA, POPO-I, POPO-I-DNA, POPO-3, propidine iodide, propidine iodide-DNA, R-Phycoerythrin pH 7.5, ReAsH, Resorufm, Resorufin pH 9.0, Rhod-2, Rhod-2Ca2+, Rhodamine, Rhodamine 110, Rhodamine 110pH 7.0, Rhodamine 123, MeOH, Rhodamine Green, Rhodamine Phalloidin pH 7.0, Rhodamine Bright Red-X Antibody Conjugate pH 8.0, Rhodamine Green pH 7.0, Rhodol Green Antibody Conjugate pH 8.0, Sapphire, SBFI-Na ⁺ , Sodium Green Na+, Thiorhodamine 101, EtOH, SYBR Green I, SYPRORuby , SYTO 13-DNA, SYTO 45-DNA, SYTOX Blue-DNA, Tetramethylrhodamine Antibody Conjugate pH 8.0, Tetramethylrhodamine Dextran pH 7.0, Texas Red-X Antibody Conjugate pH 7.2 , TO-PRO-I-DNA, TO-PRO-3-DNA, TOT O-I-DNA, TOTO-3-DNA, TRITC, X-Rhod-1Ca2 ⁺ , YO-PRO-I-DNA, YO-PRO-3-DNA, YOYO-I-DNA and YOYO-3-DNA.

In yet another embodiment, two types of fluorescently labeled oligonucleotides attached to two different particles can be used as long as the nanoparticles have the ability to quench the detectable label used. Suitable particles include polymeric particles (such as, but not limited to, polystyrene particles, polyethylene particles, acrylate and methacrylate particles), glass particles, latex particles, agarose particles, and other similar particles known in the art. Methods for attaching oligonucleotides to these particles are well known and routinely used in the art. See: Chrisey et al, 1996, Nucleic Acids Research, 24:3031-3039 (glass) and Charreyre et al, 1997 Langmuir, 13:3103-3110, Fahy et al, 1993, Nucleic Acids Research, 21:1819-1826, Elaissari et al, 1998, J.ColloidInterface ScI, 202: 251-260, Kolarova et al, 1996, Biotechniques, 20: 196-198 and Wolf et al, 1987, Nucleic Acids Research, 15: 2911-2926 (polymer/latex ).

Labels other than fluorescent molecules may be used, such as chemiluminescent molecules, which upon hybridization impart a detectable signal or a change in a detectable signal.

nanoparticles

As used herein, "nanoparticles" relate to small structures smaller than 10 microns in any direction, preferably smaller than 5 microns. In general, the nanoparticles include any compound or substance having a high loading capacity for the oligonucleotides described herein. Nanoparticles useful in the practice of the present invention include metallic (e.g., gold, silver, copper, and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetic) colloidal materials so long as these Nanoparticles have the ability to quench an otherwise detectable label. Other nanoparticles useful in _the practice of this invention include ZnS, _ZnO , _TiO2 , _AgI , AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, _In2S3 , _In2Se3 , _{Cd3P2 , Cd3As2} , InAs and GaAs. The size of the nanoparticles is preferably from about 5 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm. It is also contemplated that the size of the nanoparticles is from about 5 to about 10 nm, or from about 5 to about 20 nm, or from about 5 to about 30 nm, or from about 5 to about 40 nm, or from about 5 to about 60 nm, or from about 5 to about 70 nm, or About 5 to about 80nm, or about 5 to about 90nm, or about 5 to about 100nm, or about 5 to about 110nm, or about 5 to about 120nm, or about 5 to about 130nm, or about 5 to about 140nm, or about 10 to about 20 nm, or about 10 to about 40 nm, or about 10 to about 50 nm, or about 10 to about 60 nm, or about 10 to about 70 nm, or about 10 to about 80 nm, or about 10 to about 90 nm, or about 10 to about 100 nm, or about 10 to about 110 nm, or about 10 to about 120 nm, or about 10 to about 130 nm, or about 10 to about 140 nm, or about 10 to about 150 nm. Nanoparticles can also be rods, prisms or tetrahedrons.

Accordingly, nanoparticles are contemplated for use in the present methods, which include the use of various inorganic materials including, but not limited to, metals, semiconductor materials, or ceramics as described in US patent application No 20030147966. For example, metal nanoparticles include those described herein. Ceramic nanoparticle materials include, but are not limited to, dibasic calcium phosphate, tricalcium phosphate, alumina, silica, and zirconia. Organic materials from which nanoparticles are made include carbonaceous materials. Nanoparticle polymers include polystyrene, silicone rubber, polycarbonate, polyurethane, polypropylene, polymethylmethacrylate, polyvinyl chloride, polyester, polyether, and polyethylene. Biodegradable, biopolymers (eg peptides such as BSA, polysaccharides, etc.), other biomaterials (eg sugars) and/or polymeric compounds are also considered for use in the production of nanoparticles.

In practice, the method of the present invention provides the use of any suitable nanoparticle and its adhering molecules within the scope of detection assays generally known in the art using suitable, incompatible polynucleotide complex formation, i.e. hybridization interference, Form double-stranded or triple-stranded complexes. Particle size, shape, and chemical composition contribute to the properties of oligonucleotide-functionalized nanoparticles. These properties include, for example, optical properties, optoelectronic properties, electrochemical properties, electronic properties, stability of various solutions, magnetism and pore size changes and channels. For the use of nanoparticles having different sizes, shapes and/or chemical compositions, as well as for utilizing nanoparticles having uniform size, shape and chemical composition, mixtures of particles are contemplated for use. Examples of suitable particles include, but are not limited to, nanoparticle aggregates, isotropic (e.g., spherical particles) and anisotropic microparticles (e.g., aspheric rods, tetrahedrons, prisms) and core-shells, as proposed on December 28, 2002 US patent application Ser. No. 10/034,451 and those particles described in International application no. PCT/USO 1/50825 filed December 28, 2002, the disclosures of which are incorporated herein by reference in their entirety.

Methods of making metallic, semiconducting and magnetic nanoparticles are well known in the art. See, eg, Schmid, G. (ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, MA (ed.) Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991); Massart , R., IEEE Transactions OnMagnetics, 17, 1247 (1981); Ahmadi, TS et al, Science, 272, 1924 (1996); Henglein, A. et al, J. Phys. Chem., 99, 14129 (1995) ; Curtis, AC et al., Angew. Chem. Int. Ed. Engl, 27, 1530 (1988). Preparation of cyanoacrylate nanoparticles in Fattal, et al., J. Controlled Release (1998) 53: 137-143 and US Patent No. 4,489,055. Liu et al., in J. Am. Chem. Soc. (2004) 126:7422-7423 describe a method for making nanoparticles comprising poly(D-glucaramidoamine). Tondelli et al. describe the preparation of nanoparticles comprising polymeric methyl methacrylate (MMA) in Nucl. (1998) 26:5425-5431, and for example Kukowska-Latallo et al. in Proc. Natl. Acad. ScL USA Preparation of dendritic nanoparticles as described in (1996) 93: 4897-4902 (Starburst polyamidoaminedendrimers).

Suitable nanoparticles are also commercially available from, for example, Ted Pella, Inc. (gold), Amersham Corporation (gold), and Nanoprobes, Inc. (gold).

In addition, as also described in US patent application No20030147966, nanoparticles containing the materials described herein are commercially available, or can be obtained from progressive nucleation in solution (such as colloidal reactions), or obtained by various physical and chemical vapor deposition processes , such as sputter deposition. See, eg, HaVashi, (1987) Vac. Sci. Technol. July/August 1987, A5(4):1375-84; Hayashi, (1987) Physics Today, December 1987, pp.44-60; MRS Bulletin, January 1990, pgs. 16-47.

As further described in US patent application No 20030147966, contemplated nanoparticles are produced using _HAuCl4 and citric acid-reducing agent, using methods known in the art. See, eg, Marinakos et al, (1999) Adv. Mater. 11:34-37; Marinakos et al, (1998) Chem. Mater. 10:1214-19; Enustun & Turkevich, (1963) J. Am. Chem. Soc.85:3317. Tin oxide nanoparticles with dispersed aggregated particles having a particle size of approximately 140 nm are commercially available from Vacuum Metallurgical Co., Ltd. of Chiba, Japan. Other commercially available nanoparticles of various compositions and particle sizes can be obtained from, for example, Vector Laboratories, Inc. of Burlingame, Calif.

Nanoparticles with Structural Transformation Function

recognition sequence

In other embodiments, the detectable change is that the oligonucleotide is labeled with a molecule (eg, without limitation, a fluorescent molecule and a dye) wherein the hybridization of the oligonucleotide to the nanoparticle produces a detectable change. In one aspect, for example, an oligonucleotide or polypeptide functionalized on a nanoparticle has a label attached distal to the linking end of the nanoparticle, and in the absence of a target, the labeled The distal end is positioned close to the nanoparticle at a location sufficient to quench the fluorescence of the marker. In one aspect, metal and semiconductor nanoparticles are referred to as fluorescence quenchers, with the degree of quenching effect depending on the distance between the nanoparticles and the fluorescent molecules. Thus, in the single-stranded state, oligonucleotides attached to nanoparticles interact through the nanoparticles, for example, the formation of a hairpin structure, through the folding of the secondary structure, which allows the fluorescent molecules to be in proximity to the nanooligonucleotides. Acids make important quenching observations. Likewise, in the unbound state, the peptide attached to the nanoparticle will assume conformation, will label it with the nanoparticle, and will extinguish the label in proximity. Through the formation of nucleotide-peptide complexes, or due to the binding of target molecules through recognition sequences, fluorescent molecules will become nanoparticles with a distance that reduces fluorescence quenching (Figure 1). Useful oligonucleotide lengths can be determined empirically. Thus, various aspects of metallic and semiconducting nanoparticles have fluorescently labeled oligonucleotides attached to polypeptides or are used herein for detection in any format.

Linking oligonucleotides to nanoparticles

The nanoparticles used in the provided methods are functionalized with oligonucleotides, or modified forms thereof, that are about 5 to about 100 nucleotides in length. Methods also contemplate oligonucleotides whose oligonucleotides are from about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length Nucleotide length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to 15 nucleotides in length, about 5 to about 10 nucleotides in length The acid length, and all oligonucleotides in between, are specified to the extent that the oligonucleotide achieves the desired effect. Thus, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 nucleotide oligos Nucleotides are considered.

In other aspects, an oligonucleotide comprises from about 8 to about 80 nucleotides (ie, from about 8 to about 80 linked nucleosides). A person skilled in the art will appreciate the methods utilizing compounds 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, or 80 nucleotides in length.

Nanoparticles, oligonucleotides or both are functionalized to link oligonucleotides to nanoparticles. Such methods are known in the art. For example, oligonucleotides functionalized with thiols at their 3' or 5' ends are readily attached to gold nanoparticles. See Whitesides, 1995, Proceedings of the Robert A. Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, Tex., pages 109-121. See also, Mucic et al, 1996, Chem.Commun.555-557 (describing a method for attaching 3' thiol DNA to a flat gold surface, which can be used for attaching oligonucleotides to nanoparticles). This thiol approach can also be used to attach oligonucleotides to other metals, semiconductors and magnetic colloids with other particles listed above. Other functional groups for oligonucleotide attachment to solid surfaces, including phosphate groups (see, e.g., US Pat. No. 5,472,881 for oligonucleotide phosphate binding to gold surfaces), replacing alkylsiloxanes (see Burwell, 1974, Chemical Technology, 4:370-377 Matteucci and Caruthers, 1981, J.Am.Chem.Soc, 103:3185-3191 Oligonucleotide binding to silica and glass surfaces, and Grabar et al., Anal.Chem. , 67:735-743 binding of aminoalkylsiloxanes and similar binding of mercaptoaklylsiloxanes). Oligonucleotides terminated with 5'thionucleoside or 3'thionucleoside can also be used to attach oligonucleotides to solid surfaces. The following references describe other methods that can be used to attach oligonucleotides to nanoparticles: Nuzzo et al., 1987, J.Am.Chem.Soc, 109:2358 (gold-loaded disulfide); Allara and Nuzzo, 1985 , Langmuir, 1:45 (aluminum-supported carboxylic acid); Allara and Tompkins, 1974, J.Colloid Interface Sci., 49:410-421 (copper-supported carboxylic acid); Her, The Chemistry Of Silica, Chapter 6, (Wiley 1979) (carboxylic acid on silica); Timmons and Zisman, 1965, J.Phys.Chem., 69:984-990 (carboxylic acid on platinum); Soriaga and Hubbard, 1982, J.Am.Chem.Soc. , 104:3937 (platinum-supported aromatic ring compounds); Hubbard, 1980, Ace.Chem.Res., 13:177 (functional solvents such as sulfolanes, sulfoxides and platinum); Hickman et al., 1989, J.Am.Chem .Soc, 111:7271 (platinum isonitriles); Maoz and Sagiv, 1987, Langmuir, 3:1045 (silica carrying silane); Maozand Sagiv, 1987, Langmuir, 3:1034 (silica carrying silane); Wasserman et al al., 1989, Langmuir, 5:1074 (silane on silica); Eltekova and Eltekov, 1987, Langmuir, 3:951 (aromatic carboxylic acid, aldehyde, alcohol and methoxy groups on titanium dioxide and silica ); Lee et al., 1988, J.Phys.Chem., 92: 2597 (metal loaded rigid phosphate). In addition, any suitable method for attaching oligonucleotides to the nanoparticle surface can be used. A preferred method of attaching oligonucleotides to a surface is based on US application Ser.No.09/344,667, filed June 25, 1999; Ser.No.09/603,830, filed June 26, 2000; 2001 Ser. No. 09/760,500 filed January 12, 1997; Ser. No. 09/820,279 filed March 28, 2001; Ser. No. 09/927,777 filed August 10, 2001; and 1997 International application nos. PCT/US97/12783, filed July 21; PCT/USOO/17507, filed June 26, 2000; PCT/USO1/01190, filed January 12, 2001; March 28, 2001 described in proposed PCT/USO1/10071, the disclosure of which is incorporated herein by reference in its entirety. The aging process provides unexpectedly enhanced stability and selectivity of nanoparticle-oligonucleotide conjugates. The method involves providing an oligonucleotide covalently bonded, preferably with a moiety and a functional group thereof, that can bind to the nanoparticle. The moieties and functional groups are those that allow binding (ie chemisorption or covalent bonding, to oligonucleotides) to the nanoparticles. For example, an oligonucleotide with an alkyl disulfide bond or a cyclic disulfide bond covalently bonded to its 5′ or 3′ end can be used to bind to a different nanoparticle of nucleotides, including gold nanoparticles.

The oligonucleotides are contacted with the particles in water for sufficient time so that at least some of the oligonucleotides are bound to the nanoparticles through the functional groups. This time can be determined empirically. For example, it has been found that a period of about 12-24 hours gives good results. Other appropriate conditions for oligonucleotide binding can also be determined empirically. For example, nanoparticles at a concentration of about 10-20 nM and incubation at room temperature provide good results.

Next, at least one salt is added to the water to form a salt solution. The salt can be any suitable water-soluble salt. For example, the salt can be sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, and combinations of two or more of these salts, or one of these salts dissolved in a hydrochloric acid buffer middle. Preferably, the salt is added as a concentrated solution, but may also be added as a solid. The salt can be added to the water all at once, or gradually over time. "Gradually adding within a certain period of time" means that the salt is divided into at least two parts and added at intervals within a certain period of time. Suitable time intervals can be determined empirically.

The ionic strength of the salt solution must be sufficient to overcome electrostatic repulsion between at least some of the oligonucleotides, and electrostatic attraction between negatively charged oligonucleotides and positively charged nanoparticles or between negatively charged oligonucleotides and charged nanoparticles. Electrostatic attraction between negatively charged nanoparticles. It has been found that gradually reducing the electrostatic attraction or repulsion by gradually adding salt over a period of time maximizes the surface density of oligonucleotides on the nanoparticles. The appropriate ionic strength for each salt or combination of different salts can be determined empirically. The final concentration of sodium chloride in the phosphate buffered solution is from about 0.1 M to about 1.0 M, and it has been found that it is preferable to gradually increase the concentration of sodium chloride over a period of time to obtain good results.

After adding the salt, incubate the oligonucleotides and nanoparticles in the salt solution for an additional period of time to allow sufficient additional oligonucleotides to bind to the nanoparticles to form stable nanoparticles-oligonucleotides conjugate. As described in detail below, it has been found that the increased surface density of oligonucleotides on the nanoparticles stabilizes the conjugates. The incubation time can be determined empirically. A total incubation time of about 24-48 hours, preferably 40 hours, has been found to give good results (this is the total incubation time; as noted above, the salt concentration can be increased stepwise over this total time). The second period of incubation in the saline solution is referred to herein as the "aging" step. Other conditions for this "aging" step can also be determined empirically. For example, good results are obtained at room temperature and pH 7.0.

It has been found that conjugates prepared using the "aging" step are much more stable than those prepared without this step. As noted above, this increased stability is due to the increased oligonucleotide density on the surface of the nanoparticles obtained by the "aging" step. Another "rapid salt aging" method produced particles with comparable DNA density and stability. The salt aging step can be completed in about 1 hour when the salt is added in the presence of a surfactant, such as about 0.01% sodium dodecyl sulfate (SDS), Tween, or polyvinyl alcohol (PEG) .

The surface density obtained by the "aging" step depends on the size and type of nanoparticles and the length, sequence and concentration of oligonucleotides. The surface density that stabilizes the nanoparticles and the conditions necessary to achieve that surface density for ideal association of nanoparticles and oligonucleotides can be determined empirically. Typically, a surface density of at least 10 picomoles/ ^cm is sufficient to provide stable nanoparticle-oligonucleotide conjugates. Preferably, said surface density is at least 15 picomoles/cm ² . If the surface density is too high, the ability of the conjugate to hybridize the oligonucleotide to the nucleic acid and oligonucleotide target will decrease, so the surface density should not be greater than about 35-40 picomoles/cm ² . Also provided are oligonucleotides at least 10 picomoles/cm ² , at least 15 picomoles/cm ² , at least 20 picomoles/cm 2 , at least 25 picomoles/cm ² , at least 30 picomoles/cm ² , at least 35 picomoles/cm ² A method of binding a surface density of moles/cm ² , at least 40 picomoles/cm ² , at least 45 picomoles/cm ² , at least 50 picomoles/cm ² , or at least 50 picomoles/cm ² or higher to the nanoparticles.

Here, "hybridization" (also occasionally replaced by the term "composite formation") means that two DNAs are hydrogen-bonded according to the Watson-Crick DNA complementarity rules, Hoogstein binding rules, or other sequence-specific binding rules known in the art. Or the interaction between three nucleic acid strands. Alternatively, it refers to the interaction between the polypeptides defined herein according to the sequence-specific binding properties known in the prior art. Hybridization can be performed under different stringent conditions known in the art. Under suitable stringent conditions, the hybridization between two complementary strands or two polypeptides in the reaction can reach about 60%, about 70% or higher, about 80% or higher, about 90% or higher, about 95% % or higher, about 96% or higher, about 97% or higher, about 98% or higher, or about 99% or higher.

In various aspects, the method of the invention comprises the use of two or three oligonucleotides or polypeptides that are 100% complementary to each other, i.e. perfectly matched; or at least (meaning greater than or equal to) about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60% of the full length, At least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% are complementary to each other.

It will be appreciated by those skilled in the art that the oligonucleotide sequences used in the present methods need not be 100% complementary to each other in order to hybridize specifically. Additionally oligonucleotides may hybridize to each other at one or more segments such that intervening or adjacent segments do not participate in hybridization (eg, loop structures or hairpin structures). The percent complementarity between any given oligonucleotides can be routinely determined with the BLAST program (Basic Local Alignment Search Tools) and the PowerBLAST program known in the art (Altschul et al, 1990, J.MoI.Biol, 215:403 -410; Zhang and Madden, 1997, Genome Res., 7:649-656).

In one aspect, methods are provided wherein the packing density of oligonucleotides on the surface of nanoparticles is sufficient to result in cooperative behavior between nanoparticles and between polypeptides on individual nanoparticles. On the other hand, said cooperative behavior between nanoparticles increases the degradation resistance of oligonucleotides.

As used herein, "stable" means that the majority of the oligonucleotide remains attached to the nanoparticle for a period of at least 6 months after the conjugate is prepared and that the oligonucleotide is capable of detecting nucleic acid Hybridization to nucleic acid and oligonucleotide targets under standard conditions encountered in the method and method of nanofabrication.

Each nanoparticle used is provided in the method having a plurality of oligonucleotides attached thereto. As a result, each nanoparticle-oligonucleotide conjugate has the ability to hybridize to a second oligonucleotide that is conjugated to a fluorophore that is detectably different from the A fluorophore present on the first nanoparticle-oligonucleotide conjugate and a fluorophore functionalized on the second nanoparticle, and when present, the second oligonucleotide is a free oligonucleotide , with sufficiently complementary sequences. In one aspect, a method is provided wherein each nanoparticle is functionalized with the same oligonucleotide, ie, the oligonucleotides attached to the nanoparticles are of the same length and the same sequence. In another aspect, each nanoparticle is functionalized with two or more different oligonucleotides, i.e., at least one linked oligonucleotide differs from at least one other in terms of different length and/or different sequence. A linked oligonucleotide.

The term "oligonucleotide" or "polynucleotide" includes a single sequence attached to a nanoparticle or multiple copies of the single sequence attached. For example, in various aspects, the oligonucleotides are present in multiple copies tandem, such as two, three, four, five, six, seven, eight, nine, ten or more tandem Linked repeats.

Alternatively, the nanoparticles are functionalized to include at least two oligonucleotides with different sequences, provided that each oligonucleotide is labeled with a detectably different label. As above, the different oligonucleotide sequences are in various aspects concatenated and/or arranged in multiple copies. Alternatively, oligonucleotides with different sequences are directly attached to the nanoparticles. In methods wherein oligonucleotides having different sequences are attached to nanoparticles, aspects of the methods include wherein the different oligonucleotide sequences hybridize to different regions on the same polynucleotide.

The oligonucleotides on a nanoparticle may all have the same sequence, or may have different sequences that hybridize to different portions of another nanoparticle-linked polynucleotide. When using oligonucleotides with different sequences, each nanoparticle can have all of the different oligonucleotides attached to it, or different oligonucleotides attached to different nanoparticles. Alternatively, the oligonucleotides on the US nanoparticle may have multiple different sequences, at least one of which must hybridize to a portion of the polynucleotide on the second nanoparticle.

Nano-FLARE Technology

In one aspect of the invention, the oligonucleotide or polypeptide comprises a recognition sequence in a binding moiety that is linked to a nanoparticle as described herein. "Recognition sequence" as used herein is understood to mean partially or fully complementary to a target molecule of interest.

A nanoparticle having an attached oligonucleotide binding moiety comprising a recognition sequence is first attached to a reporter sequence. As used herein, "reporter sequence" is understood as a sequence which is partially or fully complementary and thus hybridizable to the binding moiety and its recognition sequence. As discussed above, the reporter sequence is flagged, also known as Nano-Flare. Further, the reporter sequence consists of fewer, the same, or more bases than the recognition sequence in various aspects, such that binding of the recognition sequence in the binding moiety to its target molecule causes release of the hybridized reporter sequence, resulting in ligation of the reporter sequence Detectable and measurable changes in the markers of α (Figure 2).

In a specific aspect, nanoparticles functionalized with a recognition sequence for a specific target mRNA hybridize to a reporter polynucleotide having a reporter sequence labeled with a short complementary Cy5, wherein Cy5 when hybridized to the recognition sequence on the nanoparticle Partial fluorescence quenching. This reporter sequence can also be replaced by the target mRNA. Following the displacement, the Cy5 moiety is no longer quenched and fluoresces, allowing the detection and quantification of a fluorescent signal that correlates with the amount of the target sequence, identification of the target sequence and the displacement companion with the reporter sequence sequence hybridization.

Nano-Flare exploits the unique optical properties of gold nanoparticles (Au NPs). Compared with molecular quenchers, Au NPs have higher efficiency (Dubertret et al, 2001, Nat.Biotechnol.19: 365-370) and larger distances (Dulkeith et al, 2005, Nano Lett. ) quenches the fluorescence. Likewise, all types of nanoparticles described herein can be used as long as they are capable of quenching the detectable label of the attached binding moiety.

In the case of in vitro studies, those skilled in the art will be able to determine, without undue experimentation, the relevant melting temperature and/or hybridization conditions that will facilitate binding of the reporter to the recognition sequence in the absence of the target molecule Instead, the presence of the target molecule results in displacement of the reporter sequence.

The invention is illustrated by the following examples, which are not intended to be limiting in any way.

Example

Example 1

The purpose of this example is to demonstrate that gold nanoparticle preparations modified with fluorescently labeled oligonucleotides can be used to detect intracellular molecular targets. As a proof-of-concept experiment, the use of fluorescently labeled oligonucleotide-modified gold nanoparticles is highly effective for the intracellular detection of mRNA targets in a dual cell type. These preparations readily enter cells, produce fluorescent signals, and are easily read using both fluorescence microscopy and flow cytometry.

Specifically, 13nm gold nanoparticles were modified with several different sequences, terminated with a thiol moiety at one end and a fluorescent dye at the other end, and included a structure that switches the recognition sequence. In the absence of a target, the dye molecules are in close proximity to the gold nanoparticle surface, resulting in quenching and no fluorescent signal. In the presence of the target, the dye molecules move away from the gold nanoparticle surface and a fluorescent signal is observed (Figure 1).

Au NPs function via gold-thiol linkages using thiolated oligonucleotides containing an 18-base recognition unit for specific RNA transcripts (Figure 1) (Love et al, 2005, Chem. Rev. 105: 1103-1169). Oligonucleotide-functionalized Au NPs are then hybridized with short phthalocyanine (Cy5) dyes that function as termination reporters for “flares” when replaced by long targets or target regions (Figure 1). In this bound state, the Cy5 fluorescence of the reporter chromatid is quenched due to the proximity to the Au NP surface. In the presence of the target, the flash chromatids are displaced from the Au NPs by forming long more stable duplexes in the target and oligonucleotide-modified Au NPs.

Example 2

In order to further exemplify the application of the ability of gold nanoparticles to enter cells and detect intracellular target molecules, in vitro cell culture experiments were performed.

C166 mammalian cells stably expressing enhanced green fluorescent protein were maintained in Dulbecco's Modified Eagle's Medium (37°C, 5% CO ₂ ) containing 10% serum and modified with fluorescently labeled oligonucleotides A gold nanoparticle formulation targeting enhanced green fluorescent (EGFP) protein mRNA. SKBR3 human breast carcinoma (see below) and C166 mouse endothelial cells were obtained from the American Tissue Culture Collection (ATCC) in McCoy's 5A medium containing 10% heat-inactivated live fetal bovine serum and Dulbecco's modified Eagle's, respectively. Growth medium (DMEM) was maintained at 37°C (5% CO ₂ ). Cells were seeded into 6- or 24-well plates and cultured for 1-2 days before treatment. Cells were approximately 50% confluent at the time of treatment. The medium was replaced with fresh medium containing functionalized Au NPs.

Control experiments were performed with particles containing regions targeted for anthrax RNA that are not present in mammalian cells. Sixteen hours after transfection, these EGFP-expressing cells were treated with the EGFP-targeting probe which displayed a bright fluorescent signal, very strong compared to that observed in control particles. As a further control experiment, particles were tested in C166 cells that do not express EGFP and thus do not contain the EGFP mRNA target. In these experiments, no signal was found in cells for any of the probes, so surface fluorescently labeled oligonucleotide-modified AuNP preparations can be used to detect specific intercellular molecular targets.

Probe entry into cells was confirmed using inductively coupled plasma mass spectrometry to quantify uptake and remove any sequences dependent on uptake effects. From these data it was confirmed that after a typical experiment, cells containing approximately 100,000 gold nanoparticles and C166 cells which had taken up the same number of gold nanoparticles were independent of the recognition sequences contained in the oligonucleotides.

Example 3

Further experiments were performed on fluorescently labeled oligonucleotide-modified gold nanoparticle preparations to examine their oligonucleotide loading and ability to emit fluorescent signals. The results of these experiments indicated that each gold nanoparticle functions through approximately 60 fluorescent oligonucleotides containing recognition sequences. Further, when 1 nM solutions of each oligonucleotide-modified gold nanoparticle preparation were absorbed in KCN solution, they all showed approximately the same fluorescence. Together with this characterization data, it was shown that fluorescently labeled oligonucleotide-modified gold nanoparticle formulations exhibited the same fluorescent signal in the absence of the target, further suggesting that the observed intracellular signaling was caused by specific intracellular binding conditions.

The detection of endogenous genes is very important for drug development and genetic research. Therefore, the preparation of gold nanoparticles can be used to detect the presence of the oncogene survivin. These particles again showed a bright fluorescent signal from A549 lung cancer cells expressing internal survivin when compared to the control sequence. These results indicate that the above-described fluorescently labeled oligonucleotide-modified gold nanoparticle formulations can be used to correctly read the presence of native mRNA targets.

The observed fluorescent signal can optionally be detected in a large number of processed cells using a single benchtop flow cytometer. These experiments again highlighted the high uptake efficiency observed for these fluorescently labeled oligonucleotide-modified gold nanoparticle formulations, which also showed strong signals indicative of the presence of intracellular mRNA targets in almost all samples presented. Here, 1000 cell counts were plotted as a function of their fluorescence intensity using the GuavaEasy Cyte flow cytometer and instrument software. In these experiments, it was found that a striking engraftment into a large number of A549 cells expressing survival in the fluorescence of the pigment. This result demonstrates that fluorescently labeled oligonucleotide-modified gold nanoparticle preparations are suitable for sorting large numbers of cells when coupled to flow cytometry.

Comparison of particles with conventional quencher-fluorophore oligonucleotide sequences transfected into cells using the Lipofectamine 2000 transfection reagent component was also available. Under similar conditions, the particles of the invention showed a remarkable signaling ability, with the Lipofectamine 2000 transfection reagent component showing negligible signaling ability. Even when they were concentrated 10-fold, they showed little or no signal, thus suggesting that the nanoparticles are superior to conventional quencher-fluorophore oligonucleotide probes under these conditions.

In summary, the above examples show that:

1) Gold nanoparticles facilitate intracellular expression of fluorophores containing oligonucleotides capable of detecting targets.

2) These fluorescently labeled oligonucleotide-modified gold nanoparticle formulations can be used to detect both endogenous and exogenous intracellular targets.

3) A fluorescent signal indicating the presence of a particular mRNA target can be read by either fluorescence microscopy or flow cytometry.

4) The high uptake of these preparations and their high signaling capacity make them suitable for sequencing large numbers of cells.

5) Under the conditions of this study, the high uptake of these agents and their high signaling ability outperformed conventional quencher-fluorophore oligonucleotide probes.

6) This principle can be extended to other structures, switching recognition sequences, such as nucleic acid aptamers and peptides.

According to the present invention, additional fluorescently labeled aptamers containing the target adenosine triphosphate (ATP) molecule were also investigated.

The present invention also investigates the ability to simultaneously detect multiple intracellular targets and quantify their intracellular concentrations in real time. This principle can be used for real-time monitoring of cell functions in higher organisms.

Example 4

Nano-flares have been prepared using 13nm Au NPs because particles of this size are effective quenchers and can be densely functionalized with oligonucleotides (Mirkin et ai, 1996, Nature 382:607-609) , and does not scatter visible light efficiently, which is important for designing light detection with minimal interference.

Au NPs are functionalized by gold thiol bond formation with thiolated oligonucleotides (Figure 2) containing elements that recognize specific RNA transcripts (Love et ah, 2005, Chem. Rev. 105: 1103-1169). Oligonucleotides were synthesized by the Expedite 8909 Nucleotide Synthesis System (ABI) using standard solid-phase phosphoramidite methods. The main components and reagents were purchased from Glen Research Company. Oligonucleotides were purified by reverse phase high performance liquid chromatography (HPLC). For the preparation of nanosparkle probes, citrate-stabilized gold nanoparticles (13 ± 1 nm) were prepared by published methods (Frens, G., 1973, Nature-Physical Science 241: 20-22). Thiol-modified oligonucleotides were added to 13±1 nm gold colloids at a concentration of 3 nmol oligonucleotides per ml of 10 nM colloid and stirred overnight. After 12 hours, a sodium dodecyl sulfate (SDS) solution (10%) was added to the mixture to obtain an SDS concentration of 0.1%. Phosphate buffer (0.1M; pH 7.4) was added to the mixture to obtain a phosphate concentration of 0.01M, then six aliquots of sodium chloride solution (2.0M) were added to the mixture over 8 hours to obtain a 0.15M final sodium chloride concentration. The mixture was stirred overnight to complete the functionalization process. Centrifuge (13,000 rpm, 20 min) and suspend the solution containing the functionalized particles three times in phosphate buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 niM KCl, pH 7.4, Hyclone) to produce a solution for all subsequent experiments. Purified Au NPs. The concentration of particles was determined by measuring their extinction at 524nm (ε=2.7x10L mol*cm"). In PBS (PBS; 137mM NaCl, 10niMPhosphate, 2.7mM KCl, pH 7.4, Suspend the purified oligonucleotide-functionalized AuNPs in Hyclone to a concentration of 10 nM. The mixture was heated to 70 °C, slowly cooled to room temperature, and kept in the dark for at least 12 hours for hybridization. The particles were washed with 0.2 μm acetic acid Saline rinse filters (GE Healthcare) were used for sterile filtration. The oligonucleotide sequences used were as follows:

Recognition sequence: 5′-CTT GAG AAA GGG CTG CCA AAA AA-SH-3′(SEQ ID NO.1)

Reporter sequence: 3′-CCC GAC GGT T-Cy5-5′(SEQ ID NO.2)

Target region: 3′-GAA CTC TTT CCC GAC GGT-5′(SEQ ID NO.3)

Nanoflare probes or molecular beacons were diluted to a concentration of 1 nM in PBS containing 0.1% Tween 20 (Sigma) and treated with complementary targets (target concentration, 1 μΜ). Fluorescence spectra were recorded on a JobinYvon Fluorolog FL3-22 with excitation at 633 nm and emission measured from 650 nm to 750 nm in 1 nm increments. The oligonucleotide-functionalized Au NPs are then hybridized to a short cyanine (Cy5) dye-capped reporter sequence capable of "flashing" when displaced by a longer target or region of target. In the bound state, the Cy5 fluorescence of the reporter beam is quenched due to its proximity to the Au NP surface. In the presence of the target, the flash beam is displaced and released from the Au NPs by forming longer and more stable duplexes between the target and the oligonucleotide-modified Au NPs.

Testing of the nanoflash design using synthetic complementary targets showed that detection responds to a 3.8-fold increase in fluorescent signal based on target recognition and bonding. In contrast, the signal does not change in the presence of non-complementary target and has a comparable amount relative to background fluorescence. These results demonstrate that nanoflashes are effective in signaling in the presence of specific targets.

Example 5

Investigate nanoflash detection with defined signaling capabilities and the ability of synthetic targets to enter, display and detect RNA targets in living cells. The use of nanoflares to incorporate complementary regions of the survivin transcript target has attracted considerable attention for its utility in cancer therapy and diagnosis (Altieri et al, 2003, Oncogene 22:8581-8589). The SKBR3 cell line (human breast cancer) expressing a large amount of survivin transcripts (Peng et al, 2005, Cancer Res. 65: 1909-1917) was used as a model for detection of survivin-targeted nanoflares. Survivin recognition and reporter sequences are shown above (SEQ ID NO.1 and SEQ ID NO.2). As a control, a second probe was prepared comprising non-complementary sequences. The noncomplementary probe was designed and determined to have similar background fluorescence, melting properties, and signaling capabilities as the survivin probe. The survivin control probe oligonucleotide sequence is:

Control particle recognition sequence: 5′-CTA TCG CGT ACA ATC TGC AAA AA-SH-3′(SEQ ID NO.4)

Control particle reporter sequence: 3′-GCA TGT TAG ACG T-Cy5-5′(SEQ ID NO.5)

Survivin molecular beacon: 5′-Cy 5-CGA CGG AGA AAG GGC TGC CAC GTCG dabcy 1-3′ (SEQ ID NO.6)

Control molecular beacon: 5′-Cy 5-CGA CGT CGC GTA CAA TCT GCC GTC G-dabcyl-3′ (SEQ ID NO.7).

Cells were cultured on glass microscope coverslips, incubated with nanoflares, and visualized with a scanning confocal microscope. Specifically, cells were grown on glass coverslips placed at the bottom of 6-well tissue culture plates. After 1 day, the media was replaced with media containing nanoflashes (particle concentration, 125 pM). After 6 hours of treatment, the media was replaced and the cells were cultured for an additional 12 hours. Coverslips were removed, washed with PBS, and mounted in chambers containing PBS mounted on glass slides. All images were acquired with a Zeiss 510 LSM at a magnification of 63X using a 633 nm HeNe laser excitation source.

SKBR3 cells treated with survivin nanoflares were highly fluorescent compared to cells treated with non-complementation controls. To further confirm that this signaling is consistent with the presence of survivin, the C166 cell line (murine endothelial) was used as a control since it does not contain human survivin transcripts. C166 cells were treated with survivin and control probes. In this case, no discernible difference in cell fluorescence was observed after treatment. These imaging results were consistent with reverse transcriptase PCR (RT-PCR) assays (see below).

To quantify the intracellular signaling of nanoflares, cells treated with probes were examined using resolved flow cytometry. Additionally, flow cytometry provides a means to collect fluorescence data from large populations of cells. This excludes the variability and experimental artefacts that can be observed using techniques such as fluorescence imaging that only allow the examination of small cell samples. Cells were treated with nanoflare (particle concentration, 10 nM) as described above. Molecular beacon probes (SEQ ID NO.6 and SEQ ID NO.7) were delivered to cells with Lipofectamine 2000 (Invitrogen). After treatment, the cells were detached from the culture flasks with trypsin. Flow cytometry was performed with DakoCytomation CyAn excited at 635nm.

Cell lines transfected with nanoflares showed a homogeneous single population of fluorescent cells, consistent with the greater than 99% cell invasion we observed when transfected with antisense particles (Rosi et al., Science 312:1027-1030) . Flow cytometry revealed that SKBR3 cells treated with survivin nanoflash were highly fluorescent and 2.5 times more fluorescent than the population treated with a non-complementation control. For comparison, in the C166 cell model, both probes yielded similar low fluorescent signals. These flow cytometry experiments were in excellent agreement with confocal imaging, demonstrating uniform cellular internalization and intracellular signaling by nanoflashes.

Then, experiments were performed to understand the unique properties of these probes within the context of intracellular detection experiments. First, the intracellular properties of the nanoflares were compared with those using a commercially available transfection agent (Peng et al., 2005, Cancer Res.65:1909-1917; Nitin et al., 2004, Nucleic Acids Res.32:e58) Lipofectamine Delivered molecular beacon reports compared. Molecular beacons and nanoflares were introduced into SKBR3 cells (transfection concentration, 10 pM) and their signaling abilities were investigated by flow cytometry. Cells treated with survivin nanoflash produced a 55-fold higher fluorescent signal than cells probed with survivin molecular beacons transfected at the same concentration. Fluorescence measurements outside of cell cultures showed that each nanoflare probe contains approximately 10 fluorophores and thus should have a signal 10 times greater than that of a molecular beacon at the same probe concentration. The greater-than-predicted intracellular fluorescence suggests that nanoflashes are internalized more rapidly or to a greater extent than molecular beacon detection.

Next, the molecular beacons were transfected at high concentrations (0.5 nM) to obtain intracellular fluorescent signals observable with nanoflashes. Comparison of background fluorescence (molecular beacons and nanoflashes) formed with non-complementary detection. Fluorescence detected by non-complementary molecular beacons is significantly greater than that of non-complementary nanoflashes. Because the difference between background and signal is critical for accurate target detection, lower nanoflare background provides an important advantage when detecting intracellular targets.

To examine how biochemical enzyme degradation produces non-specific signals, nanoflashes were incubated with the endonuclease DNAse I (0.38 mg/L, a concentration significantly greater than that found in the cellular environment) and detected by monitoring the increase in fluorescent signal over time. Determine the degradation ratio. Nanoflash probes were diluted to a concentration of 2.5 nM in PBS (pH 7.0), 0.25 mM MgCl2, and 50 mg/L Bovine Serum Albumin (Fischer Scientific). Bovine Pancreatic DNase I (United States Biochemical) was added immediately before reading (concentration, 0.38 mg/L). All experiments were performed with a Photal Otsuka Electronics FluoDia T70 with excitation at 620 nm and emission at 665 nm. Molecular beacons were detected in a similar manner at a concentration of 25 nM. The approximate rate of degradation under these experimental conditions was determined in the range of the linear region of the degradation curve (Rizzo et al., 2002 Molecular and Cellular Probes 16:277-283).

The results of the test showed that the nanoflares degrade at a normalized rate of 0.275 nmol mm″1 under these conditions. In contrast, the molecular beacons degrade at a normalized rate of 1.25 nmol mm″1, about 4.5 times faster than the nanoflares. Because nuclease activity produces increased background fluorescence in conventional detection, the reduced nuclease activity of NanoFlash produces a system with lower background signal.

Example 6

To illustrate the application of cell entry, enhanced signaling and low background of transcribed nano-flares with high sensitivity to alter the intracellular amount of RNA, siRNA combination experiments reduced the levels of survivin RNA transcripts in the SKBR3 cell model . When cells were about 50% confluent, siRNA anti-human survivin (Santa Curz) was expressed into cells using Lipofectamine 2000 transfection reagent (siRNA concentrations, 20, 40 and 80 nM). After 24 hours, the medium was replaced with nano-flare probes (particle concentration 50 pM). After 6 hours, the cells were washed and fresh medium was added. Cells were further cultured for 12 hours and analyzed by flow cytometry.

First, cells were treated with siRNA anti-survivin, and intracellular RNA levels were quantified using nano-flares and flow cytometry. The siRNA concentration dependence in the fluorescence of the number of cells transferred was observed as an effect of the siRNA concentration added to the cell culture medium (Fig. 3a). siRNA concentration is expressed to the left of the histogram in the graph. Of the untreated samples, half the amount exhibited equal or higher fluorescence than the mean shade of gray. In treated samples, a smaller number of cells exhibited mean fluorescence (decreasing gray, increasing black). To demonstrate that this transfer was comparable to a reduction in the number of survivin transcripts, RT-PCR measurements were performed on samples treated with the same concentration of siRNA. Counting was performed with a Guava Easy Cyte Mini (Guava Technologies). Total RNA was isolated from cells using the RNeasy Plus Kit (Qiagen) following the manufacturing protocol described below. During the lysis step, 5 x ¹⁰⁷ increased copies of green fluorescent protein (EGFP) RNA were added to each sample to account for RNA depletion during isolation and purification. To generate an RNA standard curve for qRT-PCR, the RNA fragments used for quantification are generated from appropriate RNA cells. Using PCR and a primer containing a T7 promoter site, the fragment is modified to a suitable sequence for transcription (DNA→RNA). Transcripts were purified using the MEGAclear kit (Ambion) following the manufacturer's protocol. RNA concentrations were determined with the Ribogreen RNA quantification kit (Invitrogen), and a standard curve was prepared using a dilution series of stock RNA. Primers are:

EGFP Forward 5′-TCT TCT TCA AGG ACG ACG GCA ACT-3′ (SEQ ID NO.8)

EGFP Reverse 5′-TGT GGC GGA TCT TGA AGT TCA CCT-3′ (SEQ ID NO.9)

T7EGFP Forward 5′-TGC ATA ATA CGA CTC ACT ATA GGG AGATCT TCT TCA AGG ACG ACG GGC AAC T-3′ (SEQ ID NO.10)

Survivin Forward 5′-ATG GGT GCC CCG ACG TTG-3′(SEQ ID NO.11)

Survivin Reverse 5′-AGA GGC CTC AAT CCA TGG-3′(SEQ ID NO.12)

T7 Survivin Forward 5′-TGC ATA ATA CGA CTC ACT ATA GGGAGA TGG GTG CCC CGA CGT TG-3`(SEQ ID NO.13)

Quantitative PCR and analysis were performed with the LightCycler RNA master SYBR Green Kit (Roche Applied Sciences) according to the manufacturer's recommendations. Reverse transcription was performed at 61°C for 20 minutes, followed by 45 cycles of amplification (95°C, 5 sec; 54°C, 15 sec; 72°C, 20 sec), and target gene RNA was normalized to generate a standard curve. All reactions were performed three times.

The linear decrease in fluorescence signal within the cell population was consistent with the decrease in RNA copy number present detected by RT-PCR assay (Fig. 3b). Taken together, these results suggest that Nano-Flare is sensitive to changes in the number of intracellular transcripts. RT-PCR was performed in triplicate and the error bars shown in the upper part are the standard deviation of these assays.

developed a new system of intracellular probes called "nano-flare". Nano-flares are novel and potentially very useful because they are the only probes that combine cell transfection, plum protection, RNA detection and quantification, and mRNA visualization. In addition to the uses shown in contextual siRNA knockdown experiments, nano-flares are also expected to be useful in other areas such as cell sorting, gene profiling, and real-time drug testing. Finally, given that these materials are also useful as gene regulators for their ability (Rosi et al., 2006, Science 312:1027-1030; Seferos et al., 2007, ChemBioChem 8:1230-1232), these probes are expected to be easily Ideal for simultaneous transfection, real-time control and visualization of gene expression.

Overall, these results show that:

1) Gold nanoparticles facilitate the intracellular delivery of fluorophore-containing oligonucleotides as detectable targets.

2) These fluorescently labeled modified oligonucleotide nanoparticle reagents can be used to detect, visualize and quantify intracellular targets.

3) Fluorescent letters indicating the presence and amount of specific mRNA targets can be converted into readable measurements.

4) The efficient uptake of these reagents, their high signaling capacity and low toxicity make them well suited for differentiating cell populations.

5) Under the conditions studied, the efficient uptake of these reagents and their high signaling capacity exceed conventional quencher-fluorescent oligonucleotide probes.

6) The rationale extends to other structure-switch recognition sequences such as nucleic acid aptamers and peptides.

The present invention contemplates that the probes will result in the ability to simultaneously detect multiple intracellular targets, and quantify their intracellular concentrations in real time. The principles are also envisioned for application in real-time monitoring of cellular function in higher organisms and concurrent treatment while simultaneously detecting effects.

Claims (14)

1. method of measuring the IC of target molecule, comprise target molecule and nano particle in the step that allows to contact under described target molecule and the condition that described nano particle is connected, described nano particle comprises the bound fraction that is specific to described target molecule, described bound fraction marker mark, wherein said target molecule and described nano particle be connected the detectable change that causes marker, and the intramolecular concentration of the change of wherein said detectable marker and described target molecule is proportional.

2. the method for claim 1, wherein said bound fraction is selected from the group of being made up of polynucleotide and polypeptide.

3. method as claimed in claim 2, wherein said bound fraction is DNA.

4. method as claimed in claim 2, wherein said bound fraction is RNA.

5. the method for claim 1, wherein said target molecule is selected from the group of being made up of polynucleotide and polypeptide.

6. method as claimed in claim 2, wherein said bound fraction is covalently bound polynucleotide to described nano particle, and described marker is the mark that is connected to the polynucleotide of described bound fraction multi-nucleotide hybrid, wherein said bound fraction polynucleotide and the polynucleotide that discharge described hybridization being connected of described target molecule, and after discharging, can detect described marker.

7. method as claimed in claim 6, wherein, when described polynucleotide that have described mark and the hybridization of described bound fraction, the described mark that is connected to described polynucleotide is by cancellation.

8. method as claimed in claim 2, wherein said bound fraction are the polynucleotide that contain marker, and described marker is a detectable label, and wherein said marker only can detect when described polynucleotide are connected with described tagged molecule.

9. method as claimed in claim 8, wherein, when described bound fraction was not connected with described target molecule, described marker was by cancellation.

10. method as claimed in claim 2, wherein said bound fraction is a polypeptide, and described marker is the mark that is connected with described polypeptide bound fraction, and wherein said bound fraction discharges described mark with being connected of described target molecule, and described marker can detect after discharging.

11. method as claimed in claim 10, wherein when being connected with described bound fraction, described mark is by cancellation.

12. the method for claim 1, wherein said nano particle comprises a plurality of bound fractions.

13. method as claimed in claim 12, wherein said bound fraction is connected with a target molecular specificity.

14. method as claimed in claim 12, wherein said bound fraction be connected more than a target molecular specificity.

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